Proximate composition, lipid and elemental profiling of eight varieties of avocado (Persea americana)

Eight Moroccan avocado varieties were analyzed for their nutritional composition and physicochemical properties. The nutritional contents of the sample were determined through the evaluation of the moisture, oil, ash, protein, and carbohydrate contents, and energy value calculation. Additionally, macroelements (Ca, Mg, and Na) and microelements (Fe, Zn, Cu, and Mn) were determined in the mineral profile. Oils were examined also for their fatty acid, phytosterol, and tocopherol profiles. As a result of the study, the avocado presents significant differences between the eight studied varieties (p < 0.05), with regard to moisture content (57.88 g/100 g to 84.71 g/100 g), oil content (8.41 g/100 g to 57.88 g/100 g), ash (0.57 g/100 g to 1.37 g/100 g), protein content (5.7 g/100 g to 8.61 g/100 g), carbohydrate content (5.63 g/100 g to 14.61 g/100 g), and energy value (99.9 kcal/100 g to 316.8 kcal/100 g). Sodium (5783.01 mg/kg to 12,056.19 mg/kg) was the predominant macro-element in all varieties, followed by calcium (295.95 mg/kg to 531.67 mg/kg), and magnesium (246.29 mg/kg to 339.84 mg/kg). Copper (85.92 mg/kg to 112. 31 mg/kg) was the main microelement in all varieties, followed by iron (8.5 mg/kg to 20.32 mg/kg), and manganese (7.3 mg/kg to 18.45 mg/kg), while zinc (1.72 mg/kg to 5.66 mg/kg) was detected in small amounts. In addition, significant difference was observed in lipid profiles, according to the eight studied varieties (p < 0.05). Avocado oils were mainly composed of monounsaturated fatty acids (76.89 g/100 g to 84.7 g/100 g), with oleic acid (50.38 g/100 g to 71.49 g/100 g) standing out as particularly characteristic, while β-sitosterol (l2365.58 mg/kg to 4559.27 mg/kg), and α-tocopherol (30.08 mg/kg to 182.94 mg/kg) were among its major phytosterols and tocopherols. All avocado varieties represented in this study can be consumed as a fruit as an excellent source of energy, minerals, fatty acids, phytosterols, and tocopherols. The regular consumption of this fruit provides the body with several essential nutrients.


Proximate composition
Avocado pulps powder was used for proximate composition determination.All the tests were performed in triplicate.The moisture content (MC) was determined according to ISO 665:2020 method 11 .About 5 g of pulp from each avocado variety was weighed and dried in a ventilated oven at 106 °C, until a constant weight was reached.The ash content (AC) of pulps powder was determined following ISO 18122: 2022 method 12 .The crucibles used were oven dried at 105 °C for one hour, then cooled in the desiccator to room temperature and weighed.About 5 g of pulp powder were weighed into crucibles and the whole set was put into a muffle oven where they were heated gradually.After about 1 h of heating from 0 to 250 °C, the temperature was raised to 600 °C and burning continued for another 6 h.The crucibles were removed and allowed to cool in a desiccator to room temperature before weighing.The percentage of total ash was calculated.The oil contents (OC)were carried out according to ISO 659:2009 method 13 .About 30 g of powder from each dry pulp sample was extracted by Soxhlet apparatus for 8 h, using pure hexane as solvent.The solvent was removed under reduced pressure using a vacuum a rotary evaporator and the oil was stored at −4 °C until further analysis.The extraction yield was calculated gravimetrically.The Kjeldahl method was used for the determination of the total nitrogen content 14 .The samples were digested with a mixture of H 2 SO 4 , Se and C 7 H 6 O 3 .The resulting mixture was digested for 5 h at 300 °C by using a digestion block (QBlock series, Ontario, Canada).Then the mixture was filtered and treated with 5.5 mL of the buffer solution (4 mL of Na 2 [Fe (CN) 5 NO] and 2 mL of NaClO).The mixture was kept in the dark for 15 min at 37 °C, then the absorbance of each sample was measured at 650 nm.Protein content (PC) was determined by multiplying the nitrogen content by the conversion factor of 6. 25.
Carbohydrate content (CC), and energy value (EV) were determined from protein content, moisture content, ash content, and oil content using the following equations:

Mineral determination
The concentrations of metallic elements are determined after degreasing according to the method described by Munter and Grande (1981)  15 .About 500 mg of each sample were weighed into a glass tube and dissolved in 6 mL of 70% concentrated HNO 3 .The tubes were then placed in a digestion block (QBlock series, Ontario, Canada) and the whole was heated for 60 min at 90 °C.Then 3 mL of H 2 O 2 (30%) were added to each tube and the mixture was heated at 90 °C for 15 min, then 3 mL of HCl (6 M) were added.The mineral solution in each tube was cooled and filtered and the solution was adjusted to volume of 10 mL.The concentrations of calcium, copper, iron, magnesium, manganese, sodium, and zinc were determined by inductivity coupled plasma-optical emission spectrometry (ICP-OES); (ICAP-7000 Duo, Thermo Fisher Scientific, France).

Fatty acids composition
The fatty acids composition was assessed, after transesterification to their methyl esters derivatives (FAMEs) according to ISO 12966-2:2017 and ISO 12966-4:2015 16,17 methods.The FAMEs analysis was carried out with an Agilent HP5890 gas chromatography system equipped with a flame ionization detector (FID), and a CP-Sil 88 capillary column (100 m × 250 µm i.d, 0.2 µm film thickness).The carrier gas was nitrogen, and the flow rate was 1 mL/min.The injector and detector were operated at 250 °C.The initial oven temperature was set at 155 °C for 5 min, increased to 230 °C at a rate of 1.5 °C/min, and maintained for 10 min isotherm.The injection volume was 1 µL in a split mode (1:50).The peak areas were computed by the integration software, and percentages of FAMEs were obtained by direct internal normalization as weight percentage.The standard mixture of fatty acid methyl esters (Sigma Chemical Co.) was used for the identification of the peaks.

Phytosterols composition
Phytosterol composition and content were performed according to ISO 12228-1:2014 method 18 .After a trimethylsilylation of the crude sterol fraction, the composition was carried out using an Agilent 6890 gas chromatography system equipped with a SE 54 CB column capillary column (50 m × 320 µm i.d., 0.25 µm film thickness), and coupled to flame ionization detector.The carrier gas used was helium (flow rate: 1.6 mL/min).The injector and detector temperature was 320 °C.The oven temperature was programmed from 245 to 260 °C (5 °C/min).The injection volume was 1 µL in a split mode (1:20).The results were expressed as the relative percentage of the area of each individual sterol peak to the area of all sterol peaks.Cholestan-3-ol was used as an internal standard.

Tocopherols composition
Tocopherol content and composition was determined using high-performance liquid chromatography according to ISO 9936:2016 method 19 .About, 20 µL of the filtrated solution (150 mg/mL of oil in n-heptane) was directly injected into a Merck-Hitachi low-pressure gradient system equipped with a Diol phase HPLC column (25 cm × 4.6 mm i.d.), a L-6000 pump, a fluorescence spectrophotometer (Merck-Hitachi F-1000), and a Chem-Station integration system.The mobile phase was composed of a mixture of n-heptane and tert-butyl methyl ether (99:1, V:V) with a flow rate of 1.3 mL/min.The fluorimeter excitation and emission wavelengths were 290 nm and

Plant protection statement
We complied with international guidelines for biodiversity and bioethics, specifically, the Convention on Biological Diversity (CBD) and the Nagoya Protocol on Access and Benefit-sharing.This ensures that all plant materials were collected in a way that respects the sovereignty rights of countries and Indigenous communities over their biological resources.We ensured that no plant species classified as endangered or threatened were unduly affected by our research activities.Where necessary, special care was taken to minimise impact on such species and their habitats.We declare that this study did not involve any genetically modified organisms (GMOs) and that all necessary precautions were taken to prevent any unintended release or dissemination of plant species into inappropriate environments.Furthermore, we confirm that we have followed best practices for the storage, transport, and disposal of plant materials.
The authors of this study are committed to the highest standards of research integrity and ethics, respecting biodiversity, ecosystems, and the rights of local and Indigenous communities.

Proximate composition
The assessment of the proximate composition and the nutritional value allowed a better knowledge for the farmers' perceptions of the avocado varieties in Morocco.A summary of the obtained results is illustrated in Table 1.The analysis of variance of proximate composition revealed a significant (p < 0.05) difference between tested varieties (Table 1).In this study, moisture content ranged from 57.88 g/100 g to 84.71 g/100 g and decreased as the oil content increased.Our results showed that the variety Maluma Hass had the highest moisture content value (84.71 ± 0.15 g/100 g), while its oil content was very low (8.41 ± 0.04 g/100 g).Whereas Ettinger had the lowest moisture content (57.88 ± 0.50 g/100 g), with the highest oil content (30.44 ± 0.04 g/100 g).Moisture content among tested varieties was relatively high which is in agreement with similar reported findings [20][21][22] .Moisture content constituted major part of the pulp.Many studies reported that avocado fruit is very susceptible to fungal spoilage due to the high level of moisture content 23 .Thus, low moisture content (50 g/100 g to 60 g/100 g) could delay the growth of spoilage microorganisms and enhance shelf life of avocado fruits 24 .Our outcomes revealed that oil content varied from 8.41 g/100 g (Ettinger) to 30.44 g/100 g (Maluma Hass).Oil content in Reed and Ettinger was higher than values reported by previous studies 20 .Avocado pulps ash values varied from 0.57 g/100 g to 1.37 g/100 g, with a mean of 1.01 g/100 g.Similarly, Nnaji et al. (2016)  20 , reported similar mean value that was 1.04 g/100 g. in contrast, Bora et al., 25 reported a low mean ash content in avocado pulps.
The crude protein content (N × 6.25) of the fruit pulps varied from 5.78 g/100 g to 8.61 g/100 g, Bacon had the highest level, while Zutano showed the lowest value.Interestingly, protein content level in tested varieties was higher than previous fundings 20,[26][27][28][29][30][31][32][33][34] .The total carbohydrate content ranged from 5.63 g/100 g to 14.61 g/100 g, among the studied varieties.Maluma Hass had the lowest value, while Fuerte showed the highest carbohydrate content, with a value of 14.61 ± 0.03 g/100 g.In a 100-g of avocado, the number of kilocalories ranged between 99.9 kcal/100 g (Maluma Hass) and 316.8 kcal/100 g (Ettinger), according to the studied varieties.The high energy value of avocado pulps with a low carbohydrate content suggests their use for weight loss.
The ash contents of all studied samples were quite similar to those reported by Nwaokobia et al., 35 (with a value of 1.02 g/100 g), in contrast the moisture, carbohydrate, and oil contents were different.Other studies have shown Table 1.Proximate composition (moisture content, oil content, ash content, protein content, and total carbohydrates), and energy value of avocado pulps.There is a significant difference between values in the same row that are followed by different letters (p < 0.05), according to the Tukey test.Each value is a mean of the results obtained on 2 trials ± standard deviation of this mean.MC moisture content, OC oil content, AC ash content, PC protein content, TC total carbohydrates, E energy value.www.nature.com/scientificreports/ that avocado pulp contained between 0.45 g/100 g and 1.77 g/100 g of ash content, moisture content between 67.87 g/100 g and 83.59 g/100 g, carbohydrates between 1.9 g/100 g and 8.64 g/100 g, lipids content between 6.53 g/100 g and 23.5 g/100 g, and protein between 1.20 g/100 g and 2.76 g/100 g 20,[26][27][28][29][30][31][32]34 .

Minerals determination
Minerals are required for the proper functioning of the body.Furthermore, they play an important role in protecting the immune system against infection, inflammation, and even cancer 36 .In this study, the concentrations of three macroelements (Ca, Mg, and Na), and four microelements (Fe, Zn, Cu, and Mn) were investigated.The obtained results (Table 2) showed significant difference (p < 0.05) of the mineral profile among the studied varieties.Sodium was the most dominant macro-mineral, with Maluma Hass having the highest value (12,056.19 ± 3.29 mg/kg).In addition, calcium concentration was significantly high in avocado pulp, the range was from 295.95 mg/kg to 531.67 mg/kg, with Ettinger variety showing the highest concentration, whereas Hass variety contained the lowest concentration.Calcium plays an essential role for the formation of blood clots, maintaining blood pressure, as well as contracting muscles 37,38 .As for magnesium, which was the least present macroelements, Reed variety showed the highest concentration (339.84 ± 0.22 mg/kg), whereas Zutano and Hass varieties showed slightly lower concentration 246.29 ± 1.38 mg/kg, and 247.09 ± 0.12 mg/kg.As a result, the avocado fruit can be recommended to those suffering from stress, fatigue, and anxiety.
Although microelements are present in small amounts, they are essential for all living organisms, including plants, and are involved in many physiological functions 39 .
Copper was found to have the highest concentration among microelements, with the highest content recorded in Ettinger variety (112.31 ± 0.19 mg/kg), while Bacon (86.68 ± 0.01 mg/kg), and Fuerte (85.92 ± 0.43 mg/kg) had the lowest contents.Several physiological processes rely on copper, including angiogenesis, neurohormone homeostasis, brain development, pigmentation, and immune function 40 .Iron, and Manganese were recorded almost with similar proportions.The Hass variety had the highest iron content (20.32 ± 0.25 mg/kg), while Fuerte variety showed the lowest value (8.23 ± 0.08 mg/kg).The variety Bacon had the highest Manganese content with a value of 18.45 ± 0.02 mg/kg, while the Choquette had only 7.48 ± 0,04 mg/kg.In contrast, Zinc was the underrepresented microelement, the highest concentration (5.66 ± 0.03 mg/kg) was found in the Fuerte variety, while Reed had only 1.72 ± 0.00 mg/kg.
Many studies have indicated that avocado pulp contains mineral content ranging from 17.4 mg/kg to 150 mg/ kg of calcium, 1.6 mg/kg to 1.8 mg/kg of copper, and 5 mg/kg to 20 mg/kg of Iron.The magnesium content ranges from 14 mg/kg to 600 mg/kg, the manganese content ranges from 0.3 mg/kg to 27.3 mg/kg, the sodium content ranges from 4.7 mg/kg to 150 mg/kg, and the zinc content ranges from 0.5 mg/kg to 6.8 mg/kg 28,31,34,41 .

Chemical composition of the vegetable oil of the avocado
Fatty acid composition.
Fatty acid composition was assessed after conversion to their methyl esters derivatives and analysis by GC-FID.fatty acid composition of avocado pulp oils is displayed in Table 3.The obtained results indicated a significant difference between the eight studied varieties (p < 0.05).
The avocado pulps contained a significant amount of oil, which makes it an important source for food and cosmetic industries 42 .Figure 2 illustrates the total levels of unsaturated fatty acids (USFAs), saturated fatty acids (SFAs), and polyunsaturated fatty acids (PUFAs), as well as the P/S and U/S ratios.Unsaturated fatty acids (USFAs) accounted for 76.89 g/100 g to 84.7 g/100 g of total fatty acids, in which 8.48 g/100 g to 21.43 g/100 g were polyunsaturated fatty acids, while 15.03 g/100 g to 22.91 g/100 g was monounsaturated (MUFA).The avocado pulp oil contained five unsaturated fatty acids and two saturated fatty acids.Among the seven identified fatty acids, oleic acid (C18:1) was the predominant fatty acid, its rate varied from 50.38 g/100 g (Maluma Hass) to 71.49 g/100 g (Zutano).Avocado oil can thus be regarded as an oleic oil.It has been suggested that oleic acid may have a moderate and controversial positive effect on LDL cholesterol 43,44 .Linoleic acid (C18:2) was also detected with a significant level varying from 7.79 g/100 g (Zutano), to 19.81 g/100 g (Fuerte).Two other UFAs were also identified, which are linolenic acid (C18:3) (0.54 g/100 g Bacon to 1.62 g/100 g Fuerte), and palmitoleic acid (C16:1) (2.06 g/100 g Choquette to 9.83 g/100 g Hass).Palmitic acid (C16:0) was the main saturated fatty acid, with a value ranging from 14.44 g/100 g to 22.42 g/100 g, the variety with the highest level was Maluma Table 2. Mineral profile of the eight varieties of Moroccan avocado pulp (mg/kg).There is a significant difference between values in the same row that are followed by different letters (p < 0.05), according to the Tukey test.Each value is a mean of the results obtained on 2 trials ± standard deviation of this mean.Ca calcium, Cu copper, Fe iron, Mg magnesium, Mn manganese, Na sodium, Zn zinc.www.nature.com/scientificreports/Hass, whereas Zutano presented the lowest value.In addition, stearic and arachidic acids were found in trace amounts (less than 1 g/100 g of total fatty acids) in avocado fruit pulp oils, with their contents ranging from 0.4 g/100 g to 0.85 g/100 g and 0.17 g/100 g to 0.29 g/100 g respectively.Similarly, Dubois et al. 45 found that avocado vegetable oil contains over 60 g/100 g of monounsaturated fatty acids, oleic acid was the foremost detected fatty acid (60.3 g/100 g).In contrast to our findings, they found a higher percentage of saturated fatty acids (16.4%).The main saturated fatty acid identified by Dubois et al. 45 was palmitic acid (15.7 g/100 g).There was a significant amount of polyunsaturated fatty acids (15.2 g/100 g), linoleic acid was the major compound (13.7 g/100 g).The PUFA/PFA ratio in avocado oil was higher than that in olive oil, according to Berasategi et al. 46 .Jorge et al. 47 reported that avocado oil from two varieties (Margarida and Hass) contained significant amounts of oleic acid, that was 54.72 g/100 g for the Hass variety, similarly to the same variety studied in our research, and 57.33 g/100 g for the Margarida variety, which is similar to the Fuerte variety in our study.In both varieties, Margarida and Hass, palmitic acid was 23.28 g/100 g and 19.43 g/100 g, respectively, while linoleic acid was 14.84 g/100 g and 13.22 g/100 g.

Tocopherol composition
The tocopherol content and composition of avocado oils were determined using a high-performance liquid chromatography coupled to a fluorescence detector.The obtained results revealed the presence of three tocopherols, namely α, β, and γ-tocopherols (Table 5).A significant difference was observed between the analyzed oils of the eight avocado varieties (p < 0.05).Tocopherols are known as the main antioxidant compounds, and contributes to the oil's stability 51 .Total tocopherol varied from 77.22 mg/kg to 332.18 mg/kg.Fuerte variety had the highest level (332.18 ± 0.04 mg/kg) while Bacon had the lowest value (77.22 ± 0.01 mg/kg).α-Tocopherol was the major tocopherol detected in all studied varieties, with values ranging from 30.08 ± 0.02 mg/kg (Bacon) to 182.94 ± 0.01 mg/kg (Maluma Hass).While, γ-tocopherol was detected in four varieties only, namely Choquette (52.33 ± 0.03 mg/kg), Maluma Hass (51.63 ± 0.01 mg/kg), Zutano (14.12 ± 0.01 mg/kg), and Bacon (7.87 ± 0.03 mg/kg).β-Tocopherol was only detected in Zutano with a value of 2.48 ± 0.01 mg/kg.Among the tocopherol homologs, the α-tocopherol has the highest biological potency, while γ-tocopherol was reported as the most potent antioxidant.Flores et al. (2014) 52 reported similar tocopherol content as Ettinger (40.5 mg/kg), and Zutano (103.5 mg/kg) varieties.In contrast, Ramos-Aguilar et al.(2021) 53 reported lower tocopherol contents than those found in our study for Choquette, Fuerte, Hass, Maluma Hass, and Zutano varieties cultivated in Mexico.Manaf et al.(2019) 54 found four tocopherols in oil extracted from three Indonesian avocados, including α-tocopherol (40 mg/kg to 45 mg/ kg), β-tocopherol (0.8 mg/kg to 2.0 mg/kg), γ-tocopherol (3.8 mg/kg to 4.2 mg/kg), and δ-tocopherol (0.04 mg/ kg to 0.08 mg/kg).the reported α-tocopherol concentrations were similar to those found in the variety Ettinger, β-tocopherol contents were similar to those found in Zutano, while γ-tocopherol contents were lower than our values.On the other hand, δ-tocopherol was not detected in tested avocado varieties.
The coefficient of correlation between the moisture, oil, ash, protein, carbohydrate content, energy value, and micro and macro elements in avocado pulp is presented in Fig. 3 and Supplementary Table 1.The energy value showed a significant positive correlation (r = 0.98, ***P < 0.001) with oil content and Calcium (r = 0.88, P < 0.01), whereas the energy value was negatively correlated with moisture content (-0.92,P < 00.001).Moisture content was negatively correlated with oil content (r = -0.92,P < 0.01) and Calcium (r = −0.8,P < 0.05).A significant positive correlation was obtained between Ash content and magnesium; the coefficient of correlation was r = 0.71 (P < 0.05).whereas the correlation between ash and the remaining nutrients was statistically non-significant (Fig. 4).Similarly, Protein content showed no significant correlation with other tested traits.

Conclusion
The current study investigated the nutritional value of eight varieties of avocados from Morocco.Our results revealed that avocado fruit pulps were rich in minerals (Ca, Cu, Fe, Mg, Mn, Na et Zn), protein and oil content.As a result of its high fat content, numerous properties and its many benefits for health, avocados are of great interest on the industrial level.The avocado oil is among the vegetable oils the easiest to extract.Among the fatty acids in the pulp, oleic acid (≥ 70%) is a major component, while the sterol fraction contains a high content of sterols and features β-sitosterol (≥ 80%) predominance.Analysis of the chemical composition of tocopherols indicates that α-Tocopherol is the major tocopherol.Based on the results of the present study, all avocados investigated, regardless of the variety, contain a variety of nutrients.Consequently, the pulp of these avocados needs to be valorized.

Figure 2 .
Figure 2. Total fatty acid content (%) of different varieties of avocado fruit.

Figure 3 .
Figure 3. Correlation between proximate composition, energy value and micro and macroelements of eight varieties of avocado.